Mobile phase, gradient formation

In addition to the stationary and mobile phases, separations obtained in TLC are affected by the vapor phase, which depends on the type, size, and saturation condition of the chamber during development. The interactions of these three phases as well as other factors, such as temperature and relative humidity, must be controlled to obtain reproducible TLC separations. The development process with a single (isocratic) mobile phase is complicated because of progressive equilibration between the layer and mobile phase and separation of the solvent components of the mobile phase as a result of differential interactions with the layer, which leads to the formation of an undefined but reproducible mobile phase gradient. 

The mono-, di-, and tri-phosphorylates of d4T were analysed by ion-pair LC-MS after lysis of the PBMC cell in Tris/methanol and centrifugation [50]. The supernatant was injected into the LC system with a 150x2.1-mm-ID Cjg column (5 pm) and a mobile-phase gradient of 70 to 35% solvent A (10 mmol/1 DMHA and 3 mmol/1 ammonium formate adjusted at pH 11.5) in solvent B (50% acetonitrile in 20 mmol/1 DMHA and 6 mmol/1 ammonium formate). Negative-ion ESI-MS was performed in SRM mode. The method enabled the direct measurement of the chain terminator ratio (d4T-triphosphate/deoxythymidine-triphosphate). Subsequently, the same group [51] reported modifications of this method, including simplifications of the sample pretreatment, replacement of the LC column for another type, and reduction of the column inner diameter from 2 mm ID to 0.32 mm ID. This improved method was applied to the determination of the phosphorylates of d4T, 3TC, and ddl. The sample throughput is 200 samples per week. The determination of intracellular AZT-triphosphate in PBMC [52], and the validation of the method for the determination of the ddl and d4T triphosphates was reported separately [53]. 

Mobile phase Gradient. MeCN MeOH 20 mM ammonium formate from 2.5 2.5 95 to 47.5 47.5 5 over 19 min. 

Modifications to the procedures described above have been to use a Micropak NHj-lO column with a mobile phase of cyclopentane-2-propanol (98/5 1.5) for the resolution of the perbenzoylated derivatives of NFA-glycosylceramide and NFA-galactosylceramide (Mc-Cluer and Evans, 1976). Formation of the O-acetyl-A -p-nitrobenzoyl derivatives of neutral glycosylceramides with subsequent chromatography on a silica stationary phase in combination with a mobile phase gradient system of 1-5% 2-propanol in hexane-dichloromethane (2 1) and UV detection at 254 nm has been reported (Suzuki et al., 1980). 

Jost et al. (212) studied the use of TLC as a pilot technique for transferring retention data to column LC (HPLC). TLC is potentially an inexpensive and convenient method for this purpose if essentially identical phases with the same retention mechanisms are used. However, there are inherent procedural differences in TLC and HPLC, which make exact transfer of data questionable. These differences include a capillary mobile phase driving force in TLC, and forced flow with constant and adjustable rates in HPLC formation of mobile phase gradients (solvent demixing) when multicomponent solvents are used in TLC preloading of the stationary phase with components from the gas phase of the TLC solvent and the presence of binder in layers but not columns. 

Mobile phase Gradient. MeCNrlO mM ammonium formate containing 0.1% formic acid 0 100 for 3 min, to 90 10 over 17 min, return to initial conditions over 5 min. 

Mobile phase Gradient. A was 50 mM pH 3.5 ammonium formate buffer. B was MeCN 50 mM pH 7.2 anunonium phosphate buffer 50 50. A B ratio not given. Flowrate 1 Detector UV 254, UV 280... 

Mobile phase Gradient. MeCN pH 4.00 ammonium formate buffer from 32 68 to 65-35 over 7 min. 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

In order to evaluate pump flow rate reproducibility and pulsation, one method is commonly used to assess gradient formation capability. A certain amount of an analyte with adequate molar absorptivity at the wavelength employed for detection is introduced into one of the mobile phases employed to create the gradient. In the case described, 5% acetone was introduced into the mobile phase, distributed to the system by pump B. No UV-absorbing analyte was introduced into mobile phase A. The fractional flow rate of pump B relative to the total flow rate of the system (mandated by the sum of the flow rates of pumps A and B) was increased in individual steps to account for 0, 3,6,12.5,25, 50, and 100% fractional rates. The total flow for the system was maintained at 300 /jL/ min (for 24 columns), resulting in a per column flow rate of 12.5 /iL/min/column. 

The mobile phases used to provide separations that interface cleanly with the MS are of great importance. Both isocratic and gradient elution can be used. High purity (HPLC grade) water, acetonitrile, and Ci to C4 alcohols are compatible with APTelectrospray and APCI. Less polar solvents such as hexane, cyclohexane, toluene, and ethyl acetate are also compatible with APCI. In general, it is advisable to always have an organic solvent present in the mobile phase to reduce surface tension, which enhances the formation of smaller, more uniform droplets and also aids vaporization and ionization and hence provides greater sensitivity. 

FIGURE 16 Comparison of a HILIC separation (top) and a reversed-phase separation (bottom). Peak I morphine, peak 2 morphine 3- glucuronide.Top column Atlantis HILIC Silica, 4.6x50mm, 3.0 lm gradient from 90% to 50% acetonitrile with lOmM ammonium formate buffer, pH 3.0 flow rate 2.0mL/min. Bottom Atlantis dC g, 4.6x50mm, 3.0pm mobile phase 2% acetonitrile with lOmM ammonium formate buffer, pH 3.0 flow rate l.4mL/min. 

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